Epoxyeicosatrienoic Acids Activate K+ Channels in Coronary Smooth Muscle Through a Guanine Nucleotide Binding Protein
Abstract Epoxyeicosatrienoic acids (EETs) are endothelium-derived arachidonic acid metabolites of cytochrome P450. They dilate coronary arteries, open K+ channels, and hyperpolarize vascular smooth muscles. However, the mechanisms of these smooth muscle actions remain unknown. This study examined the effects of EETs on the large-conductance Ca2+-activated K+ channel (KCa) in smooth muscle cells of small bovine coronary arteries. In cell-attached patch-clamp experiments, 11,12-EET produced a 0.5- to 10-fold increase in the activity of the KCa channels when added in concentrations of 1, 10, and 100 nmol/L. In the inside-out excised membrane patch mode, 11,12-EET was without effect on the activity of the KCa channel unless GTP (0.5 mmol/L) or GTP and ATP (1 mmol/L) were added to the bath solution. In the presence of GTP and ATP, the increase in the KCa channel activity with 11,12-EET in inside-out patches was comparable to that in cell-attached patches. This effect of 11,12-EET in inside-out patches was blocked by the addition of GDP-β-S (100 μmol/L). In outside-out patches, 11,12-EET also increased the KCa channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-GSα antibody (100 nmol/L) in the pipette solution completely blocked the activation of the KCa channels induced by 11,12-EET. An anti-Gβγ or anti-Giα antibody was without effect. We conclude that 11,12-EET activates the KCa channels by a GSα-mediated mechanism. This mechanism contributes to the effects of EETs as endothelium-derived hyperpolarizing factors to hyperpolarize and relax arterial smooth muscle.
Recent studies have indicated that coronary endothelial cells synthesize EETs, a family of cytochrome P450 epoxygenase metabolites of arachidonic acid.1 2 3 4 5 Likewise, the intima of coronary blood vessels possesses cytochrome P450 monooxygenase activity.6 7 In vitro studies have demonstrated that EETs dilate coronary arteries4 5 8 9 as well as renal, cerebral, pial, and caudal arteries.10 11 12 13 We have found that all four regioisomeric EETs relax coronary arteries in nanomolar concentrations in vitro.4 5 8 EETs activate K+ channels and hyperpolarize vascular smooth muscle in similar concentrations.8 10 11 14 These studies suggest that EETs are excellent candidates to serve as endothelium-dependent vasodilators that hyperpolarize vascular smooth muscle. In this regard, we and other investigators have reported that inhibition of cytochrome P450 blocks the endothelium-dependent vasorelaxation to arachidonic acid, whereas induction of cytochrome P450 monooxygenase enhances the vasodilation to arachidonic acid.3 7 8 9 EETs appear to mediate a portion of the endothelium-dependent relaxation to acetylcholine and bradykinin in coronary arteries.7 8 9 15 16 Cytochrome P450 inhibitors blocked acetylcholine-induced endothelium-dependent hyperpolarization and relaxation of coronary vascular smooth muscle.8 16 17 In addition, acetylcholine stimulates EET release.8 These studies led us to propose that EETs serve as EDHFs in coronary arteries.8
The mechanism by which EETs dilate coronary arteries and hyperpolarize vascular smooth muscle remains unknown. Recent studies have indicated that EETs activate a KCa channel in vascular smooth muscle cells.8 10 14 These results further support the hypothesis that EETs serve as EDHFs, since the KCa channels are thought to mediate the effect of EDHF.18 19 The purpose of the present study was to examine the effect of 11,12-EET on the activity of large-conductance KCa channels in vascular smooth muscle cells isolated from small bovine coronary arteries and to determine the mechanism by which 11,12-EET activates these channels.
Materials and Methods
Isolation of Vascular Smooth Muscle Cells From Small Coronary Arteries
Bovine hearts were obtained from a local slaughterhouse. A branch of the coronary artery was cannulated and filled with 10 to 20 mL of ice-cold 3% Evan’s blue in 50 mmol/L sodium phosphate containing 0.9% sodium chloride at pH 7.4 (PSS) and 6% albumin. Then the heart was dissected into 2×3×1-cm pieces and sliced into 300-μm-thick tissue sections. Small coronary arteries stained with Evans blue were identified under a dissecting stereomicroscope. These arteries were microdissected, pooled, and stored in ice-cold PSS. The dissected small coronary arteries were first incubated for 30 minutes at 37°C with collagenase type II (340 U/mL) (Worthington), elastase (15 U/mL) (Worthington), dithiothreitol (1 mg/mL), and soybean trypsin inhibitor (1 mg/mL) in HEPES buffer consisting of (mmol/L) NaCl 119, KCl 4.7, CaCl2 0.05, MgCl2 1, glucose 5, and HEPES 10 (pH 7.4). The digested tissue was then agitated with a glass pipette to free the vascular smooth muscle cells, and the supernatant was collected. Remaining tissue was further digested with fresh enzyme solution, and the supernatant was collected at 5-minute intervals for an additional 15 minutes. The supernatants were pooled and diluted 1:10 with HEPES buffer and stored at 4°C until used.
Single-channel K+ currents were recorded using the patch-clamp technique as described by Hamill et al.20 Cell-attached, inside-out, and outside-out configurations were used to identify the KCa channels and to determine the effect of 11,12-EET on the K+ currents in vascular smooth muscle cells. Patch pipettes were made from borosilicate glass capillaries that were pulled with a two-stage micropipette puller (PC-87, Sutter) and heat-polished with a microforge (MF-90, Narishige). The pipettes had tip resistances of 8 to 10 MΩ for single-channel recordings when filled with 145 mmol/L KCl solution. Smooth muscle cells were placed in a 1-mL perfusion chamber mounted on the stage of a Nikon inverted microscope. After the tip of the pipette was positioned on a cell, a high-resistance seal (5 to 15 GΩ) was formed between the pipette tip and the cell membrane by applying a light suction. The activity of K+ channels in the membrane spanning the pipette tip was recorded. These measurements represented the cell-attached mode. Inside-out membrane patches were excised by lifting the pipette membrane complex to the air/solution interface. Outside-out membrane patches were obtained by withdrawing the pipette tip from the cell after establishment of the whole-cell configuration, in which the membrane within the pipette was disrupted by a large pulse of suction.
An EPC-7 patch-clamp amplifier (List Biological Laboratories, Inc) was used to record single-channel currents. The amplifier output signals were filtered at 1 KHz with an eight-pole Bessel filter (Frequency Devices Inc). Currents were digitized at a sampling rate of 3 kHz and stored on the hard disk of a Gateway 486 DS66 computer for off-line analysis. Data acquisition and analysis were performed with pClamp software (version 5.7.1, Axon Instruments). Average channel activity (NPo) in patches was determined from recordings of several minutes by the following: where N is the maximal number of channels observed in conditions producing high levels of Po, T is the duration of the recording, and tj is the time with j=1,2 … N channels opening.
For single-channel recordings in the cell-attached mode, the bath solution contained (mmol/L) KCl 145, CaCl2 1.8, MgCl2 1.1, glucose 10, and HEPES 5 (pH 7.4), and the pipette solution contained (mmol/L) KCl 145, CaCl2 1.8, MgCl2 1.1, and HEPES 5 (pH 7.4). For single-channel recordings using the inside-out excised membrane patch, the bath solution contained (mmol/L) KCl 145, MgCl2 1.1, HEPES 10, and EGTA 2, along with 300 nmol/L ionized Ca2+ (pH 7.2). To determine the sensitivity of the channels to cytosolic Ca2+, the concentration of ionized Ca2+ in the bath solution was varied from 10−7 to 10−6 and then to 10−5 mol/L. Ca2+ concentration was estimated by a computer program21 and was confirmed by measuring the free Ca2+ concentration in the solution using fura 2 (Molecular Probes Co) with a dual-wavelength spectrofluorometer (Perkin-Elmer). The pipette solution contained (mmol/L) KCl 145, CaCl2 1.8, MgCl2 1.1, and HEPES 10 (pH 7.4). For single-channel recordings in the outside-out configuration, the bath solution contained (mmol/L) KCl 145, CaCl2 1.8, MgCl2 1.1, glucose 10, and HEPES 10 (pH 7.4), and the pipette solution contained (mmol/L) KCl 145, MgCl2 1.1, HEPES 10, and EGTA 2, along with 100 nmol/L ionized Ca2+ (pH 7.2). All patch-clamp experiments were performed at room temperature, ≈20°C.
Identification of the KCa Channel in Small Bovine Coronary Arteries
To establish current-voltage relations of the KCa channel, inside-out patches were exposed to symmetrical KCl (145 mmol/L) solutions, and single-channel currents were recorded while membrane potential was varied from −60 to +60 mV in steps of 20 mV. K+ selectivity of the single-channel current was determined by reducing K+ concentration in the pipette solution to 5.4 mmol/L (n=5). By changing the concentration of ionized Ca2+ from 10−7 to 10−5 mol/L on the cytosolic side of inside-out patches, the sensitivity of this KCa channel to intracellular Ca2+ concentration was examined (n=8). The effect of TEA (Sigma) (n=4) and IBX (Research Biochemicals Inc) (n=5) on single K+ channels was examined using outside-out excised membrane patches. TEA was added to bath solution at concentrations of 0.1, 0.3, and 1 mmol/L. IBX was added to bath solution at a concentration of 100 nmol/L.
Patch-Clamp Studies on the Effect of 11,12-EET
In cell-attached patches, symmetrical KCl (145 mmol/L) solutions were used to null the membrane potential of the single smooth muscle cell to near 0 mV. A 3-minute control recording at a membrane potential of +40 mV was obtained after a tight seal was established. Then the bath solution was rapidly changed by flushing the perfusion chamber with 10 mL of the same solution containing 11,12-EET (1, 10, or 100 nmol/L, n=7), 12-HETE (10 or 100 nmol/L, n=5), or 20-HETE (10 or 100 nmol/L, n=6), and a series of 3-minute recordings was obtained. To examine the interaction of cholera toxin and 11,12-EET on the activity of the KCa channel, 100 ng/mL cholera toxin was included in the pipette solution (n=8).
The excised-patch modes were used to further determine the mechanisms for the effect of 11,12-EET on the activity of the KCa channels. After inside-out patches were established, a 3-minute control recording was obtained at a membrane potential of +40 mV. Then the bath solution was rapidly changed by flushing the perfusion chamber with 5 to 10 mL of the same solution containing 1, 10, or 100 nmol/L 11,12-EET (n=6) with 0.5 mmol/L GTP and 1 mmol/L ATP, and a second successive 3-minute recording was obtained.
In some experiments, the concentration of ionized Ca2+ on the cytosolic side of inside-out patches was changed from 10−7 to 10−5 mol/L in the presence and absence of 11,12-EET (100 nmol/L), and the KCa channel current was recorded for 3 minutes at each Ca2+ concentration (n=5).
The excised inside-out patch mode was used to determine the effect of GDP-β-S (100 μmol/L) on 11,12-EET–induced activation of the K+ channel (n=6). GDP-β-S (100 μmol/L) and 11,12-EET were added to the GTP/ATP bath solution. The outside-out patch mode was used to examine the effects of anti-GSα (n=7), anti-Gβγ (n=8), and anti-Giα (n=4) antibody (New England Nuclear and Signal Transduction, Inc) and rabbit IgG (n=4). Antibodies at concentrations of 10 or 100 nmol/L were added to the pipette solution containing GTP/ATP.22
Western Blots of GSα Protein
The dissected coronary arteries were cut into very small pieces and homogenized with a glass homogenizer in ice-cold HEPES buffer containing 25 mmol/L sodium HEPES, 1 mmol/L EDTA, and 100 μmol/L phenylmethylsulfonyl fluoride. The homogenate containing 30 μg protein was incubated with 11,12-EET at concentrations of 1 nmol/L to 10 μmol/L for 30 minutes and then subjected to 12% SDS-PAGE at 200 V for 65 minutes (Bio-Rad).23 The proteins were electrophoretically transferred onto a nitrocellulose membrane. The membrane was washed and probed with a 1:1000 dilution of a specific anti-GSα antibody (New England Nuclear). The ECL detection kit (Amersham) was used to detect the specific GSα protein bands as described by the manufacturer.
Data are presented as mean±SEM. The significance of the differences in mean values between and within multiple groups was examined using an ANOVA for repeated measures, followed by a Duncan’s multiple-range test. Student’s t test was used to evaluate statistical significance of differences between two paired observations. Single-channel conductances were fit by least-squares linear regression or by using the Goldman-Hodgkin-Katz constant field equation. A value of P<.05 was considered statistically significant.
Characterization of KCa Currents in Small Bovine Coronary Arteries
The K+ channel activity was characterized using inside-out and outside-out excised membrane patches that were exposed to symmetrical KCl solutions (145 mmol/L) to enhance single-channel conductances. Unitary K+ currents were detected predominantly at membrane potentials from −60 to +60 mV in inside-out patches (Fig 1A⇓). The current-voltage relationship for this channel was linear between −60 to +60 mV, and mean slope conductance was 256.3±5 pS, with a reversal potential of ≈0 mV. When the K+ concentration in the pipette was reduced to 5.4 mmol/L, the reversal potential shifted in a manner predicted by the Nernst equation for K+. This shift in reversal potential in response to changes in K+ gradient across the membrane suggests that this channel is selective for K+ (Fig 1B⇓). This large-conductance K+ current was activated by membrane depolarization. At the resting membrane potential, the activity of this K+ channel was low, with a mean open probability (NPo) of 0.002±0.0001. When the membrane patch was depolarized to +60 mV, NPo was increased to 0.15±0.02.
The effects of changes in the cytosolic Ca2+ concentration on the activity of this channel were examined using inside-out patches. When the Ca2+ concentration on the cytosolic side of the membrane patch was increased from 10−7 to 10−5 mol/L, the activity of K+ channel increased markedly. At a cytosolic Ca2+ concentration of 10−7 mol/L and membrane potential of +40 mV, the NPo of this K+ channel was 0.02±0.0012. When cytosolic Ca2+ concentration was increased to 10−6 and then to 10−5 mol/L, the NPo of this K+ channel was increased to 0.04±0.003 and 1.88±0.06, respectively.
TEA and IBX, inhibitors of the KCa channel, were studied using the outside-out membrane patch mode. Representative tracings depicting the results of these experiments are presented in Fig 2A⇓. Addition of TEA to the bath produced a concentration-dependent reversible flickery-type blockade of the K+ channel. The mean unitary current amplitude of this channel fell from 9.89 pA under control conditions to 6.8, 4.7, and 2.24 pA after 0.1, 0.3, and 1 mmol/L TEA, respectively, was added to bath (Fig 2B⇓). NPo of this K+ channel was not altered by the addition of TEA. Fig 2C⇓ presents representative tracings depicting the effect of IBX on this K+ channel. IBX (100 nmol/L) decreased NPo of the channel by 93% when added to the bath (Fig 2D⇓), but it had no effect on the current amplitude of this channel. These data are consistent with this being a large-conductance KCa channel.
Effect of 11,12-EET on the Activity of the KCa Channel in the Cell-Attached Patch Mode
Representative recordings of single-channel K+ currents that were recorded in the cell-attached mode before and after the addition of 11,12-EET to the bath are presented in Fig 3A⇓. 11,12-EET caused a concentration-dependent increase of the activity of the KCa channel. 11,12-EET at concentrations of 1, 10, and 100 nmol/L produced a 0.5- to 10-fold increase in NPo of this KCa channel (Fig 3B⇓). A significant effect was seen even at the lowest concentration of 11,12-EET studied (1 nmol/L) (P<.05). The amplitude of these channels was unaltered by 11,12-EET even at the highest concentration studied (100 nmol/L) (Fig 3C⇓). When cell membrane potential was changed by adjusting the pipette potential from −20 to −40 and then to −60 mV, the activity of the KCa channel was significantly increased as described above, but the effects of 11,12-EET were not altered. 11,12-EET at a concentration of 100 nmol/L produced an ≈10-fold increase in NPo of the KCa channels in spite of changes in membrane potential from 20 to 60 mV. 12-HETE, a structural analogue of 11,12-EET, had no effect on the activity of the KCa channel, and 20-HETE decreased the activity of this KCa channel (P<.05) (Table 1⇓).
Effect of 11,12-EET on the Activity of the KCa Channel in the Inside-out Patch Mode
In contrast to the marked effects of 11,12-EET (100 nmol/L) in cell-attached patches, 11,12-EET had no effect on the activity of the KCa channel when applied to the internal surface of inside-out excised membrane patches (Table 2⇓). The number of channel openings, mean open time, and the amplitude of these KCa channels recorded from inside-out excised membrane patches were not significantly altered when even a high concentration of 11,12-EET (1 μmol/L) was applied to the internal surface of the patch. NPo was 0.03±0.009 for control and 0.0267±0.01 with 11,12-EET. However, when 0.5 mmol/L GTP and 1 mmol/L ATP were included in the bath solution, 11,12-EET produced a concentration-dependent increase in the KCa activity (Fig 4A⇓). 11,12-EET increased the NPo of these channels to an extent comparable to that in the cell-attached patch mode and in comparable concentrations. The increase in the channel activity was reversed by washing out the 11,12-EET (Fig 4B⇓). In addition, addition of 0.5 mmol/L GTP alone also increased the basal activity of the KCa channels by 15% and restored the effect of 11,12-EET in these excised membrane patches to a level comparable to that obtained in the cell-attached patches (Table 2⇓). However, ATP alone had no effect on the basal activity of the KCa channels, and 11,12-EET did not stimulate the KCa channels in the presence of only ATP.
In the presence of GTP and ATP in the bath solution, the activity of the KCa channel was also increased in response to the increase in intracellular Ca2+ concentration. NPo of the KCa channel was 0.044±0.0013, 0.2995±0.04, and 1.88±0.02 at cytosolic Ca2+ concentrations of 10−7, 10−6, and 10−5 mol/L, respectively. When 11,12-EET was added to the bath, Ca2+-induced increase in NPo of the KCa channel was not altered. NPo of the KCa channel was 0.275±0.02, 0.832±0.12, and 2.61±0.2 at Ca2+ concentrations of 10−7, 10−6, and 10−5 mol/L, respectively. Calculated pCa50 in the presence of 11,12-EET averaged 2.3×10−6 mol/L, which was not significantly different from 2.6×10−6 mol/L in the absence of 11,12-EET in the bath solution.
Effect of GDP-β-S on 11,12-EET–Induced Activation of the KCa Channel in the Inside-out Patch Mode
11,12-EET at a concentration of 100 nmol/L produced a 6-fold increase in NPo of the KCa channels in the presence of GTP/ATP. GDP-β-S reduced NPo of the KCa channels by 16% and completely abolished the effect of 11,12-EET on the activity of these channels (Fig 5A⇓).
Effect of Anti-GSα Antibody on 11,12-EET–Induced Increase in the KCa Channel Activity in the Outside-out Patch Mode
Addition of 11,12-EET to the bath solution had no effect in the excised outside-out patches in the absence of GTP and ATP in the cytosolic solution: NPo was 0.15±0.02 in the control condition and 0.167±0.023 with 100 nmol/L EET. When GTP and ATP were included in the pipette solution, 11,12-EET increased markedly the activity of these KCa channels. NPo of the KCa channels was increased from 0.15±0.02 to 0.52±0.01, but channel amplitude was not altered when 11,12-EET (100 nmol/L) was added to the bath.
The effects of antibodies against GSα, Giα, or Gβγ on the response of the KCa channel to 11,12-EET were examined using this outside-out patch mode. When an antibody against GSα at a concentration of 100 nmol/L was included in the pipette solution, addition of 11,12-EET (100 nmol/L) to the bath failed to alter the activity of the KCa channels. This inhibitory effect of the anti-GSα antibody was lost when the antibody concentration was decreased to 10 nmol/L or when it was boiled for 10 minutes. When an anti-Gβγ at a concentration of 100 nmol/L, which completely blocks the stimulatory effect of purified Gβγ subunits on the KCa channels (data not shown), was substituted for anti-GSα antibody in the pipette solution, 11,12-EET still increased the activity of the KCa channel to a comparable extent. Anti-Gi antibody or rabbit IgG (100 nmol/L) had no effect on 11,12-EET–induced increase in the KCa channel activity. Changes in NPo of the KCa channels induced by 11,12-EET in the presence of different antibodies in the pipette solution are summarized in Fig 5B⇑. Only anti-GSα antibody inhibited the increase in NPo of the KCa channel induced by 11,12-EET.
Effect of Cholera Toxin on the Activity of the KCa Channel
Cholera toxin at a concentration of 100 ng/mL increased the activity of the KCa channels by 6-fold (Fig 6A⇓). In the presence of cholera toxin in the pipette solution, 11,12-EET did not further increase the KCa channel activity. NPo of the KCa channel was 0.1033±0.03 and 0.125±0.01 before and after the addition of 11,12-EET in the bath solution, respectively (Fig 6B⇓). Cholera toxin did not alter the current amplitude of the KCa channels (Fig 6C⇓).
Presence of GSα in Coronary Smooth Muscle Cells
Based on molecular weight and reaction with a specific anti-GSα antibody, there is a smooth muscle cell protein that appears to be the α subunit of GS. Western blot of the smooth muscle homogenate with anti-GSα antibody gave two protein bands of ≈52 and 45 kD. Similar results were obtained with the GSα standard. 11,12-EET had no effect on the electrophoretic migration of GSα.
In the present study, the K+ channel activity was characterized in vascular smooth muscle cells isolated from small bovine coronary arteries using the patch-clamp technique, and the effects of 11,12-EET on the activity of K+ channels were examined. Using single-channel recording modes, we found that the dominant K+ channel in vascular smooth muscle cells isolated from small bovine coronary arteries was the large-conductance (256.3-pS) KCa channel. This finding is consistent with previous reports that the KCa channel is the most active channel found in vascular smooth muscle cells isolated from the coronary artery of the rabbit and dog.24 25
Recent studies have indicated that the vasodilatory response to EETs is associated with activation of the KCa channels in vascular smooth muscle cells isolated from cat cerebral arteries, rabbit portal vein, rat caudal arteries, and guinea pig aorta.10 14 In these studies, vascular smooth muscle cells were isolated from large arteries, and EET concentrations of 0.3 to 10 μmol/L were required to activate these channels. In the present study, using the cell-attached mode for single-channel recording, we found that the addition of 11,12-EET markedly enhanced the frequency of opening of the KCa channel in vascular smooth muscle cells in concentrations as low as 1 nmol/L. These data suggest that vascular smooth muscle cells from small coronary arteries are more sensitive to the effect of EET. In this regard, we have recently found that smaller vessels are more sensitive than large vessels to the vasorelaxant effect of EETs in isolated bovine coronary arterial rings.8 Why vascular smooth muscle cells isolated from small coronary arteries are more sensitive to 11,12-EET than those obtained from other species and vascular beds remains to be determined. This may reflect species differences or differences in vascular reactivity between vascular beds. Another possibility is that vascular smooth muscle cells from small resistance arteries like those studied in the present study are inherently more sensitive to the stimulatory effect of 11,12-EET on the KCa channels.
EETs are a series of metabolites of arachidonic acid synthesized by a cytochrome P450 epoxygenase.26 Although the regiospecific effect of the EETs was not determined in the present study, previous studies have demonstrated that the four regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) have similar stimulatory effects on the activity of the KCa channels in vascular smooth muscle cells isolated from coronary and caudal arteries and aorta.8 14 As a result, we studied only 11,12-EET as a prototype. However, 12-HETE, a structural analogue of 11,12-EET, and 20-HETE, another cytochrome P450 metabolite of arachidonic acid, did not activate the KCa channels. In contrast, 20-HETE markedly reduced the activity of the KCa channels. These results indicate that the epoxide group of the EETs is essential for activation of the KCa channels.
To determine the mechanism by which 11,12-EET increases the activity of the KCa channels in smooth muscle cells, excised-membrane patches were used to examine the effects of 11,12-EET on the activity of the KCa channels. In contrast to the results obtained in cell-attached patch mode, 11,12-EET had no effect on the activity of the KCa channel when added to the cytoplasmic surface of excised inside-out or outside-out membrane patches. These results suggest that 11,12-EET does not directly act on the KCa channels but that some cytosolic component or second messenger system is required to alter the KCa channel activity. These results are consistent with previous reports of others.11 14
To further determine the cellular mechanism mediating the effect of 11,12-EET on the KCa channels, a series of experiments was performed using excised-membrane patches to examine whether the stimulatory effect of 11,12-EET on the KCa channel could be reconstituted. Surprisingly, when GTP alone or GTP and ATP were included in the cytosolic solution in inside-out or outside-out patches, the effect of 11,12-EET on the KCa channel activity was completely restored. This finding suggested that 11,12-EET–induced activation of the KCa channel is a GTP-dependent mechanism and may involve GTP binding proteins. By using the inside-out patch mode, the effect of a GTP binding protein inhibitor, GDP-β-S, was examined on 11,12-EET–induced activation of the KCa channels. GDP-β-S abolished the effect of 11,12-EET on the activity of the KCa channels. These findings further suggest that GTP and a GTP binding protein may contribute to 11,12-EET-induced activation of the KCa channel.
Since the GTP binding protein GS has been reported to participate in the regulation of the KCa channel activity,27 28 activation of GS may be involved in the effect of 11,12-EET on the activity of the KCa channel. To test this hypothesis, an anti-GSα antibody was used to block GS activity. Addition of an anti-GSα antibody to the cytosolic solution blocked the effect of 11,12-EET on the activity of the KCa channel. Since 11,12-EET was added to the external cell membrane surface and the antibody to the cytosolic surface, it is unlikely that blockade was due to a direct binding of 11,12-EET to the antibody. In addition, the addition of an anti-Giα antibody, anti-Gβγ antibody, or rabbit IgG to the cytosolic solution in the pipette had no effect on activation of the KCa channels by 11,12-EET. Therefore, we conclude that a specific blockade of GS abolishes the effect of 11,12-EET on the activity of the KCa channels. Our findings support the view that 11,12-EET activates a G protein, likely GS, and, subsequently, the KCa channels. Since only anti-GSα antibody blocked 11,12-EET–induced activation of the KCa channels, the data imply that GSα subunit is implicated in the effect of EET.
GSα may regulate the KCa channel via two independent mechanisms.29 GSα can activate adenylyl cyclase and promote cAMP accumulation, resulting in PKA-dependent phosphorylation of the channel or some proteins coupled to the channel.30 31 32 Alternatively, the GSα may have a direct action on the channel or a closely associated protein.28 With respect to the cAMP-PKA–dependent mechanism, an increase in the production of cAMP in response to 11,12-EET would be required. However, in a recent study, we found that the vasodilator effect of 11,12-EET is not associated with an increase in the tissue content of cAMP and cGMP.8 Furthermore, ATP would be required for PKA to phosphorylate the channel. However, GTP alone can restore the effect of 11,12-EET on the channel, so ATP is not required. These data suggest that the cAMP-PKA–dependent mechanism is not involved. Therefore, the direct action of GSα on the KCa channel is a likely mechanism for the effect of 11,12-EET. It has been reported that this membrane-delimited action of GSα is a ubiquitous mechanism for regulating K+ and Ca2+ channels.33 34 Although the significance of this action of GSα in the gating of the KCa channels has not been characterized, previous studies on the regulation of Ca2+ channels have indicated that this membrane-delimited pathway for GSα is far faster (<1 second) than the cytoplasmic cAMP pathway (≈30 seconds).35 36 This membrane action of GSα has been shown to enhance the effect of agonists on ion channels.37 38 However, the detailed coupling mechanisms between GSα and the KCa channels for this membrane-delimited pathway are not yet known. GSα might act upon the channel proteins either directly, without requiring any other membrane component, or indirectly, via intermediate membrane-associated effectors. Further elucidation of the coupling mechanism between GSα and the KCa channel in coronary vascular smooth muscle will require functional reconstitution of the interacting proteins in an artificial membrane system. On the other hand, our results cannot exclude the possibility that second messengers independent of cAMP and cGMP may participate in the regulation of the activity of the KCa channel or mediate the effect of 11,12-EET in the intact cells. This dual modulation of the KCa channel through membrane-delimited action of GS and some cytosolic signaling molecules has been reported by others.28 29
There is evidence for a high-affinity binding site for 14(R),15(S)-EET in guinea pig mononuclear cell membranes, suggesting that there may be EET receptors.39 Whether there is a specific receptor for 11,12-EET in the vascular smooth muscle cell membrane remains to be determined. However, our results raise questions about this possibility, since addition of 11,12-EET to the cytosolic solution in excised inside-out membrane patches also increased the activity of the KCa channels in the presence of ATP and GTP. It is, however, possible that 11,12-EET could diffuse across the membrane and activate a receptor on the external surface. Nevertheless, the findings that the effects of 11,12-EET on the activity of the KCa channels were reversible in the excised patches but not in the cell-attached patches suggest that 11,12-EET may activate GSα on the cytosolic side of the membrane and that GSα may be a target for the effect of lipid-soluble mediators that are slowly washed out of intact cells. This direct effect of 11,12-EET on the GSα activity independent of the receptors on the cell surface may represent a unique mechanism mediating the action of lipid-soluble mediators in vascular smooth muscle cells, by which activation of GSα only produces a membrane-delimited effect but does not stimulate the production of cAMP. Alternatively, 11,12-EET may have a second action in intact cells that is not observed in inside-out patches. This second action must be slowly reversible. A recent study has indicated that EETs stimulated the endogenous ADP-ribosylation of a 52-kD protein in the liver.40 It remains to be determined whether this endogenous ADP-ribosylation is activated by 11,12-EET in coronary arterial smooth muscle and what the relationship is between EET-activated endogenous ADP-ribosylation and the activity of GS.
In summary, 11,12-EET stimulates activation of the KCa channel in small bovine coronary arteries. This increase in the activity of the KCa channel appears to be due to activation of a GTP binding protein, probably GS. Activation of the KCa channel appears to contribute to the vasodilator effect of 11,12-EET by hyperpolarizing the vascular smooth muscle.8 EETs represent EDHFs.
Selected Abbreviations and Acronyms
|EDHF||=||endothelium-derived hyperpolarization factor|
|KCa channel||=||Ca2+-activated K+ channel|
|NPo||=||open-state probability of N channels|
|PKA||=||protein kinase A|
This study was supported by grants from the National Heart, Lung, and Blood Institute (HL-51055 and HL-57244) and the American Heart Association, Wisconsin Affiliate, Inc (95-GB-52). The authors thank Gretchen Barg for her secretarial assistance and Phillip F. Pratt and Dr William S. Edgemond for providing the 11,12-EET.
Reprint requests to Pin-Lan Li, MD, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
- Received July 8, 1996.
- Accepted March 27, 1997.
- © 1997 American Heart Association, Inc.
Revtyak GE, Hughes MJ, Johnson AR, Campbell WB. Histamine stimulation of prostaglandin and HETE synthesis in human endothelial cells. Am J Physiol. 1988;255:C214-C225.
Pfister SL, Falck JR, Campbell WB. Enhanced synthesis of epoxyeicosatrienoic acids by cholesterol-fed rabbit aorta. Am J Physiol. 1991;261:H843-H852.
Rosolowsky M, Falck JR, Willerson JT, Campbell WB. Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries. Circ Res. 1990;66:608-621.
Rosolowsky M, Campbell WB. Role of PGI2 and EETs in the relaxation of bovine coronary arteries to arachidonic acid. Am J Physiol. 1993;264:H327-H335.
Pinto A, Abraham NG, Mullane KM. Arachidonic acid-induced endothelial-dependent relaxation of canine coronary arteries: contribution of a cytochrome P-450 dependent pathway. J Pharmacol Exp Ther. 1987;240:856-863.
Campbell WB, Gebremedhin D, Pratt PF, Harder DR. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res. 1996;78:415-423.
Gebremedhin D, Ma Y-H, Roman RJ, VanRollins M, Harder DR. Cellular mechanism of cerebral epoxyeicosatrienoic acid on cerebral arterial smooth muscle. Am J Physiol. 1992;263: H519-H525.
Zou A-P, Fleming JT, Falck JR, Jacobs ER, Gebremedhin D, Harder DR, Roman RJ. Regional and stereospecific effects of 11,12-epoxyeicosatrienoic acids on vascular tone and K+ channel activity in renal arterioles. Hypertension. 1994;24:382. Abstract.
Carroll MA, Garcia MP, Falck JR, McGiff JC. 5,6-Epoxyeicosatrienoic acid, a novel arachidonic metabolite: mechanism of vasoactivity in the rat. Circ Res. 1990;67:1082-1088.
Proctor KG, Falck JR, Capdevila J. Intestinal vasodilation by epoxyeicosatrienoic acids: arachidonic acid metabolites produced by a cytochrome P450 monooxygenase. Circ Res. 1987;60:50-59.
Pfister SL, Campbell WB. Arachidonic acid and acetylcholine-induced relaxation of rabbit aorta. Hypertension. 1992;20:682-689.
Singer HA, Peach MJ. Endothelium-dependent relaxation of rabbit aorta, I: relaxation stimulated by arachidonic acid. J Pharmacol Exp Ther. 1983;226:790-795.
Van de Voorde J, Vanheel B, Leusen I. Endothelium-dependent relaxation and hyperpolarization in aorta from control and renal hypertensive rats. Circ Res. 1992;70:1-8.
Godt RE. Calcium-activated tension of skinned muscle fibers of the frog. J Gen Physiol. 1974;63:722-739.
Codina J, Yatani A, Grenet D, Brown AM, Birnbaumer L. The α subunit of the GTP binding protein GK opens atrial potassium channels. Science. 1987;236:442-445.
Leblanc N, Wan X, Leung PM. Physiological role of Ca2+ activated and voltage-dependent K+ currents in rabbit coronary myocytes. Am J Physiol. 1994;266:C1523-C1537.
Falck JR, Schueler VJ, Jacobson HR, Siddhanta AK, Pramanik B, Capdevila J. Arachidonate epoxygenase: identification of epoxyeicosatrienoic acids in rabbit kidney J Lipid Res. 1987;28:840-846.
Kume H, Graziano MP, Kotlikoff MI. Stimulatory and inhibitory regulation of calcium-activated potassium channels by guanine nucleotide-binding proteins. Proc Natl Acad Sci U S A. 1992;89: 11051-11055.
Scornik FS, Codina J, Birnbaumer L, Toro L. Modulation of coronary smooth muscle KCa channels by Gsα independent of phosphorylation by protein kinase A. Am J Physiol. 1993;265: H1460-H1465.
Nelson MT, Quayle JM. Physiological role and properties of potassium channels in arterial smooth muscle. Am J Physiol. 1995;268:C799-C822.
Sadoshima J, Alkaike N, Tomoike H. Ca-activated K+ channel in cultured smooth muscle cells of rat aorta media. Am J Physiol. 1988;255:H410-H418.
Kume H, Kotlikoff MI. Muscarinic inhibition of single KCa channels in smooth muscle cells by a pertussis-sensitive G protein. Am J Physiol. 1991;261:C1204-C1209.
McDonald TF, Pelzer S, Trautwein W, Pelzer DJ. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev. 1994;74:365-507.
Kozlowski RZ, Twist VW, Brown AM, Powell T. Flash photolysis of intracellular caged GTP gamma S increases L-typed Ca2+ currents in cardiac myocytes. Am J Physiol. 1991;216:H1665-H1670.
Yatani A, Brown AM. Rapid beta-adrenergic modulation of cardiac calcium channel currents by a fast G protein pathway. Science. 1989;245:71-74.
Pelzer S, Shuba YM, Asai T, Codina J, Birnbaumer L, McDonald TF, Pelzer D. Membrane-delimited stimulation of heart cell calcium current by beta-adrenergic signal-transducing GS protein. Am J Physiol. 1990;259:H264-H267.
Wong PY, Lin KT, Yan YT, Ahern D, Iles J, Shen SY, Bhatt RK, Falck JR. 14(R),15(S)-Epoxyeicosatrienoic acid (14(R),15(S)- EET) receptor in guinea pig mononuclear cell membranes. J Lipid Mediat Cell Signal. 1993;6:199-208.
Seki K, Hirai A, Noda M, Tamura Y, Kato I, Yoshida S. Epoxyeicosatrienoic acid stimulates ADP-ribosylation of a 52 KDa protein in rat liver cytosol. Biochem J. 1992;281:185-190.